White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. They are also involved in allergies/asthma due to the development of an unwanted or excessive type of immune response to substances (antigens) in our environment and in autoimmune diseases. Because T cells respond to foreign substances (antigens) in the form of peptide- major histocompatibility complex (MHC) molecule pairs on cell surfaces, we wish to know how such complexes interact with specific receptors to evoke the effector activities of mature T cells in the body, as well as regulate their growth, inactivation, or death. In particular, we want to understand in molecular detail the protein-protein interactions that turn recognition of antigen by T cells into signals guiding the normal survival and effector functions of these cells, how variations in these recognition and signaling events leads to desirable versus undesirable forms of immunity, and how we can manipulate these events to augment useful immune responses and inhibit damaging ones. Our studies currently focus on biochemical regulatory pathways that help T cells discriminate between self- and foreign peptide:MHC molecule complexes, on the explicit mathematical modeling of these signaling processes in eukaryotic cells, and on how the antigen receptors (TCR) of T lymphocytes are selected to provide maximally effective responses to infectious agents. We previously reported our analysis of the molecular details of two novel regulatory pathways controlling early signaling by the T cell receptor (SHP-1 phosphatase dependent negative control and MAPK mediated positive control) and a full mathematical computer description of T cell receptor (TCR) signaling that incorporates these novel regulatory pathways. This year we placed our several studies in this area in the context of emerging information on circuit control in biological systems and reviewed how these data provide new insights into the operation of the adaptive immune system. In the course of these studies, we also discovered that several hours after TCR stimulation most T cells begin to express the negative regulatory transcription factor Foxp3 that limits the extent and duration of cytokine production. A detailed analysis of the conditions leading to accumulation vs. extinction of Foxp3 expression in CD4+ T cells revealed that there is a major locus of control of expression of this protein at the post-transcriptional (protein) level. Our data reveal that Foxp3 is an intrinsically unstable protein highly dependent on molecular chaperones or partner protein binding for resistance to aggregation and degradation. Elevated temperature (fever), and a combination of strong TCR and CD28 stimulation that produces many client proteins for Hsp90 in the cytosol, limit access of Foxp3 to such stabilizing partners, resulting in its aggregation and degradation. Because Foxp3 positively regulates its own gene transcription, processes that limit accumulation of Foxp3 at the protein level have an amplified inhibitory effect by also reducing Foxp3 gene activity. Single cell studies showed a direct relationship in individual cells between the level of Foxp3 and cytokine gene responses. Thus, our results show that Foxp3 can serve as a cell autonomous regulator of effector function tuned to the host environment. Steady-state conditions in which there is normal temperature, weak self ligand stimulation and low costimulation support Foxp3 expression that can interfere with autoreactive responses, whereas during infections, a combination of fever, strong foreign antigen stimulation, and strong costimulatory molecule expression suppress this regulatory loop by preventing Foxp3 protein accumulation, allowing effector responses that are needed by the host. Over the past year we have focused on using the level of CD5 expression to monitor the affinity of a specific TCR for self-MHC ligands. Using a variety of methods including analysis of partial TCR-associated zeta-chain phosphorylation, we found that we could readily classify T cells according to their self-ligand affinity by this means. Using new methods for assessing the interaction of naive T cells with foreign antigen ligands in the form of oligomers of foreign peptide-MHC class II molecule complexes (pMHC tetramers), we have made the remarkable finding that the affinity of TCRs for self-ligand as determined in the thymus during positive selection directly correlates with the affinity of the T cell for foreign pMHC. In addition, in comparison to other cell surface proteins, CD5 (whose level of expression marks self-pMHC affinity of a T cell) is skewed to the right compared to a log normal distribution. These results indicate that thymic positive selection operates to maximize the affinity of the cells selected into the mature pool, up the to limit set by negative selection, and provide a strong evolutionary rationale for the process of thymic positive selection - maximizing the ability of the mature T cell pool to recognize and respond to foreign antigens. These findings are surprising in light of several decades of work on the structural basis of T cell pMHC recognition and the role of peptides vs. MHC molecules in thymic selection and peripheral antigen recognition. Our observations indicate that the field will need to re-visit the interpretations placed on crystal structures of TCR-pMHC interactions to explain the self-foreign antigen affinity connection we have uncovered. These data also have important implications for understanding the nature of T cells responding to self antigens that promote autoimmune disease and for identifying the best subset of cells to use in adoptive immunotherapy. We have also conducted experiments combining flow cytometric studies with intravital 2 photon imaging to assess the role of self-recognition in naive lymphocyte trafficking through lymph nodes. Our data reveal that we can quantitate clear differences in the length of CD4 T cell interactions with MHC class II+ vs. MHC class II - dendritic cells and that this is correlated with a difference in the time it takes naive CD4 T cells to transit through the lymph node of wild-type and MHC class II-deficient mice. These studies have also unexpectedly revealed a systematic difference in the length of time CD4 vs. CD8 T cells spend in a lymph node. These studies provide additional evidence for non-negligible interactions between the TCRs on naive T cells and ambient self-ligands in the normal host, interactions of sufficient magnitude to affect the dynamics of lymphoid populations with lymphoid tissue.